OBJECTIVETo investigate the mechanism of Paeoniflorin (PF) in vitro to illustrate its neuroprotective effects of ameliorating cerebral ischemic/reperfusion injury which might be mediated by adenosine A1 receptor.METHODS In HEK293 cells stably transfected with A1 R (HEK293/A1 R) and primary cultured rat cortical neurons, we treated serum-starved cells with PF or pretreated with several inhibitors before PF treatment, and then lysed cells to detect downstream signaling pathways by Western Blot.Receptor interactions were investigated by co-immunoprecipitation and immunofiuorescence assays.Oxygen glucose deprivation and reperfusion (OGD/R) model was applied to study survivability of neurons assessed by MTT assay.RESULTS In HEK293/A1 R cells, PF activated PI3K/Akt and MAPK/ERK signaling pathways beginning at 5 min while EGFR (Tyr 1068) was also phosphorylated.A1 R antagonist DPCPX and selective Epidermal Growth Factor Receptor (EGFR) kinase inhibitor AG1478 could abolish ERK1/2 and Akt phosphorylation which inferred that A1 R and EGFR would be involved in signaling cascade.Furthermore, A1R and EGFR co-localized on the surface of HEK293/A1 R cells.In HEK293/A1R cells, inhibitor of Matrix Metalloprotease (MMP) BiPs but not inhibitor of Src kinase PP2 could inhibit ERK1/2 phosphorylation induced by PF.In cultured cortical neurons, PF protected neurons from OGD/R injury while it phosphorylated both Akt and ERK1/2.CONCLUSION Taken together, PF could promote cultured neurons survival in OGD/R injury and the mechanism of its neuroprotective effect was that PF transactivated EGFR signaling cascade which activated both PI3K/Akt and ERK1/2 signaling pathways mediated by A1R.